A confocal laser microscope is configured such that laser light is focused on a specimen through an objective lens, a light flux of reflected light, scattered light, or fluorescent light generated on the specimen is transmitted by an optical system, and the light flux transmitted through a pinhole disposed at an optically conjugated position with respect to a light focusing point on the specimen is received on a detector. Disposing the pinhole makes it possible to filter the light generated on the specimen other than the light focusing point. Therefore, the confocal laser microscope is operable to acquire an image with a good S/N ratio.
Further, the confocal laser microscope is configured to acquire a planar image of the specimen by scanning the specimen with laser light along two directions (X-direction and Y-direction) orthogonal to each other, along a plane perpendicular to the optical axis. On the other hand, the confocal laser microscope is configured to acquire a plurality of tomographic images (Z-stack images) in Z-direction by changing the distance in the optical axis direction (Z-direction) between the objective lens and the specimen, whereby a three-dimensional image of the specimen is formed.
In observing a biospecimen, it is often the case that the biospecimen is observed through a cover glass in a state in which the biospecimen is immersed in a broth. Further, generally, the objective lens is designed so that the imaging performance at a position immediately below the cover glass is best. In observing the inside of a biospecimen, it is necessary to acquire an image transmitted through a broth or biological tissues and having a certain depth at an observation position. Aberrations are generated in proportion to the distance from the position immediately below the cover glass to the observation position, and as a result, the resolution may be lowered.
Aberrations which may be generated are described in detail referring to FIG. 1A and FIG. 1B. FIG. 1A and FIG. 1B are diagrams schematically illustrating aberrations generated depending on the depth of a specimen to be observed. To simplify the description, the objective lens is designed to be optimized in observing a medium having a uniform refractive index. FIG. 1A illustrates a light flux 100 in observing a medium having a uniform refractive index, as used in the design. FIG. 1A illustrates that the light flux 100 is focused on one point without aberrations. Contrary to the above, FIG. 1B illustrates a light flux 110 in observing the surface of a specimen at the depth D. The light flux 110 is refracted on an interface 111 between the medium in contact with the objective lens and the specimen. The light flux 110 is refracted, and is not focused on one point due to the generated aberrations.
For instance, when the objective lens is a dry lens, the space between the objective lens and the specimen is filled with air. Therefore, the refractive index of the medium (air) between the objective lens and the specimen is 1.0, which is different from the refractive index of a biospecimen (e.g. 1.39). Aberrations are generated in proportion to the difference between the refractive index of the medium between the objective lens and the specimen, and the refractive index of the biospecimen, and to the depth of the biospecimen with respect to the observation position. On the other hand, when the objective lens is a water immersion lens, the space between the objective lens and the specimen is filled with water. Therefore, the refractive index of the medium (water) between the objective lens and the specimen is 1.333, which is closer to the refractive index of the biospecimen than the refractive index of air. The water immersion lens is suitable for observing a deep part of the biospecimen. However, the refractive index of the biospecimen is not equal to the refractive index of water. Therefore, aberrations are generated due to a difference between the refractive index of the biospecimen and the refractive index of water. Thus, lowering of the resolution is still a problem.
Further, the cover glasses have variations in the thickness thereof within the tolerance range from the design value (e.g. 0.17 mm). Aberrations are generated in proportion to a difference between the actual thickness of the cover glass and the design thickness due to a difference between the refractive index (=1.525) of the cover glass and the refractive index (=1.38 to 1.39) of the biospecimen. Spherical aberration having a phase distribution symmetrical with respect to an optical axis is generated due to deviations from the design value as described above.
Next, aberrations generated with respect to an off-axis light flux are described. The objective lens is designed so that the imaging performance with respect to an off-axis light flux is also best at a position immediately below the cover glass, as well as an on-axis light flux. When the inside of a specimen is observed as described above, asymmetrical aberrations as represented by coma aberration are generated in proportion to the depth of the specimen. Further, the amount of asymmetrical aberrations increases in proportion to the magnitude of the viewing angle. Therefore, when a periphery of an image obtained by photographing a specimen is mainly observed, the resolution of the peripheral portion of the image may be lowered due to asymmetrical aberrations, because the image of the specimen is obtained by a light flux having a large viewing angle.
Further, when aberrations are corrected by an aberration correction device, generally, the pupil position of the objective lens lies in the objective lens system. Therefore, positional deviation may occur in the optical axis direction between the aberration correction device and the objective lens, as far as a measure such as disposing the aberration correction device in the objective lens, or disposing the aberration correction device at such a position that the aberration correction device is conjugated with respect to the pupil position of the objective lens with use of a relay optical system, is not taken.
FIG. 2 is a diagram for representing positional deviation in the optical axis direction when a specimen is observed. In FIG. 2, to simplify the description, an aberration correction device 3 and an objective lens 4 are disposed to align with each other along the optical axis. In this example, the distance from the pupil position of the objective lens 4 to the aberration correction device 3 is Z. A light flux 200 focused on the optical axis is indicated by the solid line, and a light flux 210 converged at a position away from the optical axis is indicated by the dotted line. Out of a light flux incident on the aberration correction device 3, the off-axis light flux 210 is obliquely incident. As a result, the incident position of the off-axis light flux 210 is deviated from the incident position of the on-axis light flux 200 in a direction away from the optical axis by the distance d on a plane orthogonal to the optical axis of the aberration correction device 3. Therefore, it is necessary to consider the positional deviation when aberrations of the light flux 210 are corrected by the aberration correction device 3.
The positional deviation (distance d) in a direction orthogonal to the optical axis also occurs due to an alignment error between the objective lens and the aberration correction device, in addition to the above factor. Aberrations resulting from the positional deviation turn into asymmetrical aberrations such as coma aberration, as a difference component of spherical aberration.
As one means for solving image quality deterioration resulting from aberrations as described above, a correction ring has been proposed. The correction ring is a ring-shaped rotating member provided in an objective lens. The distances between lens groups constituting the objective lens are changed by rotating the correction ring. Aberrations due to an error in the thickness of the cover glass or observing a deep part of the biospecimen are cancelled by rotating the correction ring. A scale is marked on the correction ring. For instance, rough numerical values such as 0, 0.17, and 0.23 are indicated concerning the thickness of the cover glass. Adjusting the scale of the correction ring in accordance with the thickness of an actually used cover glass makes it possible to adjust the distances between lens groups in such a manner as to optimize the distances in accordance with the thickness of the cover glass (e.g. see Patent Literature 1).
Further, a technique of compensating for generated aberrations by a wave front conversion element is also known. This technique is a matrix-drivable shape variable mirror element that is disposed on an optical path of a microscope, a wave front is modulated by the shape variable mirror element based on wave front conversion data measured in advance, and the modulated light wave is allowed to be incident on a specimen, whereby an aberration-corrected image with a high imaging performance is acquired (see e.g. Patent Literature 2).
As the wave front conversion element, a shape variable mirror element configured such that the shape of a reflection surface thereof is electrically controllable is used. When a plane wave is incident on the shape variable mirror element, and if the shape variable mirror element has a concave shape, the incident plane wave is converted into a concave wave front (the amplitude of a concave shape is doubled).
Further, a microscope control method for controlling an aberration correction amount based on the distance between an objective lens and a specimen using such correction means is also known (see e.g. Patent Literature 3).